Project Summary In both type 1 and type 2 diabetes, hepatic secretion of glucose is a primary contributor to hyperglycemia. In turn, hyperglycemia is the root of many chronic complications associated with these diseases. Our recent in vivo data using transgenic mice confirm the potential of activating human liver pyruvate kinase (hL-PYK) to counteract the hyperglycemia associated with diabetes. To facilitate future allosteric drug designs to activate (or prevent inhibition of) hL-PYK in the treatment of hyperglycemia, the long-range goal of our laboratory is to characterize the molecular/atomic level mechanism of allosteric activation of hL-PYK by (Fru-1,6-BP), allosteric inhibition by alanine, and inhibition b protein phosphorylation. However, drugs that resemble native effectors or other biological compounds are likely to also bind other proteins and cause side-effects. As an alternative, we will now characterize areas of the hL-PYK protein outside of native effector binding sites that have roles in allosteric mechanisms and, thus, can be targeted in future rational design of allosteric drugs that do not resemble biological compounds. A primary innovation in our approach to studying allosteric mechanisms is recognition of the potential of divorce allosteric regulation from ligand binding. Therefore, we will distinguish which ligand-induced changes in the protein contribute to ligand binding and which play roles in allosteric regulations. In Aim 1 w will monitor and probe to identify which protein-substrate interactions have roles in allosteric functions, using difference attenuated total reflectance infrared spectroscopy (ATR-IR) to monitor bond changes in the substrate (we will NOT evaluate protein with IR), a substrate analogue series, a divalent metal cation series, site-directed random mutagenesis of substrate contact residues, mutant cycle analysis, and loop modifications. Effector binding will be monitored using 2nd derivative absorption spectroscopy. In turn, the binding of effector will be evaluated over a concentration range of substrate or a substrate analogue as a means of determining allosteric coupling. In Aim 2, we will use hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) and X-ray crystallography to evaluate conformational and dynamic changes among the various enzyme complexes that define the allosteric energy cycles for each regulation (Fru-1,6-BP, alanine, and phosphorylation). In Aim 3, we will initiate a study to generate hybrid tetramers that will allow us to isolate pairwise interactions between one active site and one allosteric site. This approach will be used to answer if the allosteric site in one subunit regulates the active site in the same subunit, a result that will simplify mechanistic interpretation of structural changes. Detailed understanding of the molecular mechanisms of hL-PYK will facilitate future drug designs to activate (or prevent inhibition of) this enzyme to reduced hepatic glucose secretion and counteract hyperglycemia.